With the structural analysis of DNA proceeding at a rapid pace based on the advances in DNA sequencing techniques and encouraged by potential applications of information from completely sequenced genomes of important organisms, a new chemical horizon is the synthesis of large DNA structures. This endeavor follows the trend of past chemical work on molecules of all types; first, determination of the structure, then synthesis of the structure for confirmation and producing novel structures and analogs to investigate their properties. A number of DNA analogs have been synthesized for special purposes (peptide backbone DNA, DNA with modified phosphoryl groups) and special sequence segments have been designed to interact with double helical DNA. In the case of microbial genomes, there has been discussion of preparing a minimal genome. However, general methods for constructing large precisely designed DNA segments have not been developed.
Presently, commercially available oligonucleotide synthesis can routinely produce molecules on the order of a hundred nucleotides, and through PCR amplification of known segments of a genome, defined fragments of up to 40 kilobase pairs can be prepared. Through cleavage with specific restriction enzymes and joining by ligation, designed DNA molecules (e.g., large vectors) have been made. However, this method becomes complicated as larger fragments with more restriction sites are used and each molecule to be made must have its unique route of synthesis depending on its particular arrangement of restriction endonuclease sites. The protection of certain sites by specific methylases and the recent discovery of several very rare cleaving endonucleases have extended the range of manipulations available from this basic approach.
Cloning techniques have been used to isolate and propagate large fragments on special vectors (BAC, YACs) and homologous DNA recombination has allowed the reconstruction of known chromosomal regions of over a hundred kilobase pairs. Improved systems for direct recombination have made functional studies of genes easier through “gene knockout technology” [4]. However, the defined assembly of a novel DNA sequence of large size has not been carried out. In order to produce large designed segments that are composed of DNA sections not normally together (or not even from the same organism), new methodologies need to be developed.
Potential uses for these techniques are in areas such as the analysis of function of large interrupted coding regions that exist in many human genes, and in the construction of gene sets involved in complex metabolic processes [2]. These techniques could allow for more extensive genetic reprogramming of microbes for optimal production processes (metabolic engineering) and proposals for large scale “editing” of known genomes have been made based on engineering optimization considerations [3]. Methods for the generation of such DNA would allow the formation of optimized strains for industry and provides a way to explore global structural effects in the function of microbial genomes.